As a disease caused by purine metabolism disorder, gout is recorded at the 5th century B.C. by Hippocrates of Cos (Emmerson B T, N Engl J Med, 1996, 334; 445-451), with clinical character of hyperuricemia and gouty deposition derived from the sedimentation of urates in subcutaeous, joint, and kidney. Uric acid, the final product of purine metabolism in human bodies, may cause hyperuricemia at the concentration of over 70 mg/L; and 5%-12% of patients with hyperuricemia will eventually suffer from gout. Gouty arthritis is caused by sodium urate with saturated concentration in blood or bursa mucosa, wherein the sodium urate forms microcrystal (Nancy J G et al., Arthritis Res Ther. 2006; 8(1), R12). As time passing, chronic hyperuricemia may also produce crystal of uric acid sediments around joints, soft tissues as well as certain kinds of organs; thereby causing gouty acute arthritis, gouty deposition chronic arthritis and joint deformity. Meanwhile, kidney damage is considered to be the second common clinical manifestation of gout. Chronic hyperuricemia will gradually lead to urate sediment in medulla, kidney tubule and renal interstitium, thereby provoking local inflammation, namely chronic urate nephropathy. Patients suffered from severe hyperuricemia (such as tumors, expecially leukemia and lymphoma) may have great amount of uric acid sediments in collecting duct, pelvis, calices and ureter of the kidney in a short period of time, which will lead to lumen blockage, unuresis and acute kidney failure (also called hyperuricemic nephropathy) (Hershfield M S. Cecil, Textbook of Medicine (20th), 1508-1515). Incomplete treatment of the above disease may further provoke the concurrence of gouty coronary heart disease, hyperlipidemia, etc.
With changes in diets and living habits in recent years, the uptake of high-protein and high-purine food is increased, along with the amount of gout patients, whose number has grown twice by the last 20 years (Jones D P et al., Med Pediatr Oncol, 1990, 18: 283-286). In a survey on gout patients around the Huangpu River (in Shanghai) in 1998, the incidence rate of hyperuricemia has grown to 10.1%, and that of gout to 0.34%, which is similar with the gout incidence rate in America of last 80s to 90s (0.275%-1.000%) (Chen S et al., Clin Med J., 1998, 111 (3): 228-230). Patients with hyperuricemia only need to control their diets if no clinical symptom occurs; however, with clinical symptom caused by hyperuricemia, medication will be necessary. Tools for conventional clinical therapy include: anti-inflammatory and analgesic drugs, such as colchicine, buprofen, naproxen, etc., which can control cute episode of gouty arthritis and eliminate local pain, swelling and inflammation of joint; uricosuric agents promoting the excretion of uric acid (invalid for lowered kidney function), such as probenicid, sulfinpyrazone, benzbromarone, etc.; as well as uric acid synthesis inhibitors, such as allopurinol. Allopurinol is most commonly used in treating patients suffering from gouty deposition, kidney insufficiency, leukemia and some genetic diseases, wherein it can inhibit xanthine oxidase and disable the transformation of hypoxanthine and xanthine into uric acid, and be oxidated in vivo to oxipurinol that is soluble and can be expelled with urine. However, chronic gout patients with gouty deposition can hardly be cured by all kinds of routine treatment. Additionally, after long-term uptake of the above medicines, patients will inevitably exhibit complications such as neutropenia, impaired heart function, liver and kidney dysfunction, digestive system stimulation, glycosuria caused by aplastic anemia, as well as gout, etc.
Human hyperuricemia is relevant to uricase gene mutation in the human evolution, wherein a terminator codon is induced in advance (Wu X et al., Proc. Natl. Acad. Sci. USA, 1989, 86: 9412-9416), thus disabling the human ability to synthesize active uricase and terminating the human purine catabolism at uric acid (Wu X et al., J. Mol. Evol., 1992, 34: 78-84). Urate with lower solubility (about 11 mg/100 mL water) can be transformed to more soluble allantoin (about 147 mg/100 mL water) by active uricase in the liver peroxisome of non-human primates and other mammals, hence being more effectively excreted by kidney (Wortmann R L, Kelley W N, Kelley's Textbook of Rheumatology (6 th), 2001: 1339-1376). In western countries, uricase prepared by Aspergillus flavus (Uricozyme) have been used in treating tumor chemotherapy-related hyperuricemia for more than 10 years (Zittoun R et al., Ann. Med. Interne., 1978, 127: 479-482). ELITEK, a drug of recombinant Aspergillus flavus uricase produced by Saccharomyces cerevisiae fermentation in Sanofi Corp., France, has been granted by FDA in 2002 and been used in the short-term treatment of severe hyperuricemia from tumor chemotherapy (Pui C H et al., Leukemia, 1997, 11: 1813-1816.). Meanwhile, ELITEK has been proved to be able to reduce the volume of gouty deposition after injection (Potaux L et al., Nouv. Presse. Med., 1975, 4: 1109-1112).
Uricase (EC 1.7.3.3) exists extensively in microorganisms (such as Bacillus fastidiosus, Candida mycoderma and Aspergillus flavus), plants (such as beans and chickpeas), and animals (such as pigs, cows, dogs, and papios) (Suzuki K et al., J. Biosci. Bioeng., 2004, 98: 153-158). It can catalyse the oxidation of uric acid to allantoin at the presence of oxygen, releasing carbon dioxide (Retailleau P et al., Acta. Cryst. D., 2004, 60: 453-462).
The active uricase is a tetramer protein with identical subunits, each having molecular weight of about 34 kD and consisting of 301-304 amino acids. Uricase has maximum enzymatic activity at pH 8.0 (Bayol A et al., Biophys. Chem., 1995, 54: 229-235). Among all origins, uricase has the highest activity from Aspergillus flavus, which is up to 27 IU/mg; the second highest from Bacillus fastidiosus with 13 IU/mg (Huang S H et al., Eur. J. Biochem., 2004, 271: 517-523). Additionally, bean-origined uricases have activities of merely 2-6 IU/mg. As for recombinant expressed mammal uricases, the activity of pig uricase can reach 5 IU/mg, and papio uricase only 1 IU/mg (Michael H et al., 2006, U.S. Pat. No. 7,056,713B1); while human uricase has no activity.
Studies on recombinant uricase for human application are mainly focused on high activities of microorganism uricases and low immunogenicities of mammal uricases. However, Aspergillus flavus uricase shares less than 40% of homology with hypothetic human uricase (Lee C C et al., Science, 1988, 239: 1288-1291), and easily provokes antibody from human body. Therefore, the effect of Aspergillus flavus uricase is weakened rapidly and severe anaphylactic reaction is initiated, making it impossible for long-term treatment. Although the human uricase gene is disabled by mutation, the immunogenicity of the enzyme would be reduced if the gene was reformed and the activity recovered. However, because of missense mutations accumulated during the evolution, it is difficult to recover the human uricase activity by amino acid mutations.
Among many patent publications and literatures about mammal uricase, pig-papio chimeric uricase is studied by Duke University and Savient Corp. (Michael H et al., 2006, U.S. Pat. No. 7,056,713B1), wherein 1-9 arginines is substituted with lysines in the full-length pig uricase sequence while retaining the activity, to facilitate the following PEG modification. The PEG-ylated pig-papio chimeric uricase achieves lower immunogenicity in human bodies.
Provided herein is another novel mammal uricase with one part of human uricase amino acid sequence introduced at the C-terminal, wherein the mammal uricase exhibits lower immunogenicity while retaining the enzymatic activity. A series of studies have proved that the mammal uricase and mutants thereof have improved stability in vivo and are suitable in drug compositions for the treatment of hyperuricemia-related diseases.